1. Field of the Invention
The present invention relates to compositions comprising a lipophilic cationic organometallic complex and an agent that decreases the intramembrane potential in a cell and methods whereby these compositions may be used in vitro and in vivo. The reduction in intramembrane potential results in enhancing the cellular accumulation of the lipophilic cationic organometallic compounds.
2. Description of the Background Art
Hexakis (alkylisonitrile) technetium (I) complexes are a class of low valence technetium (.sup.99m Tc) coordination compounds empirically designed as clinical myocardial perfusion imaging agents (Jones, A. G. et al. Int. J. Nucl. Med. Biol. 11:225-234 (1984), Holman, B. L., et al., J. Nucl. Med. 25:1350-1355 (1984), Holman, B. L., et al., ibid 28:13-18 (1987), Sporn, V., Clin. Nucl. Med. 13:77-81 (1988)). Conceived to be used in a manner similar to thallous chloride for the noninvasive evaluation of coronary artery disease, the compounds exploit the more favorable emission characteristics of .sup.99m Tc for applications in clinical imaging (Strauss, H. W., et al., Radiology 160:577-584(1986), Deutsch, E., et al., Science 214:85-86(1981)). Chemical analysis of these complexes with the ground state .sup.99 Tc isotope shows them to be monovalent cations with a central Tc(I) core octahedrally surrounded by six identical ligands coordinated through the isonitrile carbon. The terminal alkyl groups, when bound to the technetium, encase the metal with a sphere of lipophilicity (Jones, A. G., et al., Int. J. Nuc. Med. Biol. 11:225-234 (1984), Mousa, S. A., et al., J. Nuc. Med. 28:1351-1357 (1987)).
While the complex has proven highly successful as a clinical flow tracer (Holman, B. L., et al., J. Nucl. Med. 25:1350-1355 (1984); Wacker, F. J., et al., J. Nucl. Med. 30:301-311 (1989)), evidence has demonstrated a component of myocardial localization dependent on tissue viability (Piwnica-Worms, D., et al., J. Nucl. Med. 31:464-472 (1990); Rocco, T. P., et al., J. Am. Col. Card. 14:1678-1684 (1989); Sinusas, A. J., et al., J. Nucl. Med. 30:756 (1989)).
In the course of investigating mechanisms of cellular retention of these agents, studies demonstrated that neither the lipophilic properties nor the cationic charge alone were sufficient to characterize the uptake properties of these complexes (Piwnica-Worms, D., et al., Invest. Radiol. 24:25-29 (1989)). The requirement of lipophilicity and cationic charge for myocardial localization raised the possibility that their cellular uptake and retention mechanisms are in part determined by mitochondrial and plasma membrane potentials in a manner analogous to several other known permeant cationic probes of membrane potential (Deutsch, C. J., et al., J. Cell. Phys. 99:79-93 (1979); Litchtshtein, D., et al., Proc. Nat'l. Acad. Sci. U.S.A. 76:650-654 (1979); Bussolati, O., et al., Biochim. et Biophys. Acta 854:240-250 (1986); Akerman, K. E., et al., ibid 546:341-347 (1979); Johnson, L. V., et al., Proc. Nat'l. Acad. Sci. U.S.A. 77:990-994 (1980); Johnson, L. V., et al., J. Cell. Biol. 88:526-535 (1981); Davis, S., et al., J. Biol. Chem. 260:13844-13850 (1985)).
Subsequent studies of cellular uptake of hexakis(methoxyisobutylisonitrile) technetium, a member of the isonitrile class of coordination compounds, suggested that the uptake of the compound was affected by alterations in the plasma and mitochondrial membrane potentials (Delmon-Moingeon, L. I. et al., Cancer Res. 50:2198 (1990); Chiu, M. L., et al., J. Nucl. Med.: 31:1646-1653 (1990)).
Data from whole organ cardiac preparations also provide indirect evidence for this model of cellular uptake. In perfused rabbit heart, oaubain (1.5.times.10.sup.-6 M) and hypoxia alter net tissue extraction of Tc-MIBI (Meerdink, D. J., et al., J. Nucl. Med. 30:1500-1506 (1989)). In perfused rat hearts, metabolic inhibition with sodium cyanide (10 mM) blocks 50% of Tc-MIBI accumulation while membrane disruption with Triton X100 (0.5%) inhibits 86% of net uptake (Beanlands, R., et al., Circ. 80(S II):545 (1989)). In sum, the data are consistent with a membrane transport process for Tc-MIBI, like other non-metallic lipophilic cations, involving a non-carrier-mediated translocation and passive distribution of the agent in response to an imposed transmembrane potential.
Other uptake models for hexakis (alky isonitrile) technetium complexes have been proposed. Simple lipid partitioning was initially thought to be the sole mechanism of localization. In this context, lipophilicity was found to correlate well with cellular uptake studies and imaging intensity in vivo for the more lipophilic agents developed early in this class (Piwnica-Worms, D., et al., Invest. Radiol. 24:25-29 (1989)), although exceptions to the trend indicated other factors were involved. Alternatively, binding of Tc-MIBI (an agent of intermediate lipophilicity) to an 8-10 KDalton cytosolic protein has been proposed (Mousa, S. A., et al., J. Nucl. Med. 27:p995 (1986)).
In evaluating the relative merit of these models, a novel prediction of the potential-dependent uptake mechanism for Tc-MIBI is the augmentation of uptake kinetics by lipophilic anions. In human lymphocytes (Deutsch, C. J., et al., J. Cell. Physiol. 99:79-94 (1979)), for example, the kinetics of plasma membrane translocation of tetraphenylphosphonium (TPB), another well characterized permeant cationic probe of membrane potential, are augmented by the presence in the incubation buffer of the lipophilic anion tetraphenylborate (TPB). In addition, low concentrations of TPB increase uptake kinetics of other lipophilic cations into isolated heart mitochondria (Bakeeva, L. E., et al., Biochim. Biophys. Acta 216:13-21 (1970)), vesicles prepared from E. coli (Altendorf, K., et al., J. Biol. Chem. 250:1405-1412 (1975)), and isolated perfused rat liver (Neef, C., et al., Biochemical Pharm. 33:3991-4002 (1984)).